Healthy Offspring from Freeze-Dried Mouse Spermatozoa Held on the International Space Station for 9 Months

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months Sayaka Wakayamaa,1, Yuko Kamadab, Kaori Yamanakac, Takashi Kohdad, Hiromi Suzukie, Toru Shimazue, Motoki N. Tadaf, Ikuko Osadaf, Aiko Nagamatsug, Satoshi Kamimurab, Hiroaki Nagatomoa,h, Eiji Mizutanib, Fumitoshi Ishinod, Sachiko Yanog, and Teruhiko Wakayamaa,b,1 aAdvanced Biotechnology Center, University of Yamanashi, Yamanashi 400-8510, Japan; bFaculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan; cDepartment of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; dDepartment of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan; eDepartment of Science and Applications, Japan Space Forum, Tsukuba 305-8505, Japan; fJapan Manned Space Systems Corporation, Tokyo 100-0004, Japan; gJapan Aerospace Exploration Agency, Tsukuba 305-8505, Japan; and hCenter of Community Promotion Center, University of Yamanashi, Yamanashi 400-8510, Japan Edited by George E. Seidel, Colorado State University, Fort Collins, CO, and approved April 20, 2017 (received for review January 26, 2017) If humans ever start to live permanently in space, assisted repro- well as in other animals, and only a few papers have been pub- ductive technology using preserved spermatozoa will be impor- lished (13–18). Those studies and our previous study (19) have tant for producing offspring; however, radiation on the International suggested that mammalian reproduction in space under condi- Space Station (ISS) is more than 100 times stronger than that on tions of microgravity cannot be easily compared with reproduc- Earth, and irradiation causes DNA damage in cells and gametes. tion in other species. Here we examined the effect of space radiation on freeze-dried Another difference between space and Earth is the high level mouse spermatozoa held on the ISS for 9 mo at –95 °C, with launch of radiation in space. The doses received inside the ISS depend on sunspot cycles and cosmic rays. The average dose rate mea- and recovery at room temperature. DNA damage to the sperma- ∼ tozoa and male pronuclei was slightly increased, but the fertiliza- sured at the ISS is 0.5 mSv/day, roughly 100-fold higher than that measured on Earth (20). Ground-based studies have dem- tion and birth rates were similar to those of controls. Next- onstrated the deleterious effects of radiation on living organisms, generation sequencing showed only minor genomic differences including the induction of mutations and tumor formation between offspring derived from space-preserved spermatozoa (21, 22). Humans living for several generations in space habitats or and controls, and all offspring grew to adulthood and had normal traveling to Mars will encounter much higher cosmic radiation, fertility. Thus, we demonstrate that although space radiation can possibly putting them at high risk for cancer (23). If space radia- damage sperm DNA, it does not affect the production of viable tion also causes DNA damage to cryopreserved spermatozoa, it offspring after at least 9 mo of storage on the ISS. might lead to serious problems, such as embryo death or abortion, or cause mutations in offspring and subsequent generations. International Space Station | preservation | freeze-dry | spermatozoa | Salamander and medaka fish eggs could be fertilized and de- fertilization veloped normally during orbital space flight (6, 24), suggesting that space radiation from brief space flights does not affect fer- ince the “space dog” Laika (Лайка) was first placed into tilization or later embryogenesis in these vertebrates. However, Sorbit in 1957 (1), many humans and animals have been to mammalian oocytes are known to have a strong potential for space or stayed on the International Space Station (ISS) for more than 6 mo. In the future, humans likely will live on large- Significance scale space stations or in other space habitats for several years or even over many generations. At that time, assisted reproductive Radiation on the International Space Station (ISS) is more than technology (ART) likely will be used to produce humans in space 100 times stronger than at the Earth’s surface, and at levels habitats, given that the use of ART by infertile couples has in- that can cause DNA damage in somatic cell nuclei. The damage creased year by year and that ART can be performed with cry- to offspring caused by this irradiation in germ cells has not opreserved spermatozoa or embryos (2, 3). In a similar way, been examined, however. Here we preserved mouse sperma- domestic animals likely will be generated by artificial insemina- tozoa on the ISS for 9 mo. Although sperm DNA was slightly tion (AI) in space, because many domestic animals are already damaged during space preservation, it could be repaired by the produced by AI using long-term cryopreserved spermatozoa (4). oocyte cytoplasm and did not impair the birth rate or normality In addition, genetic diversity is very important for maintaining a of the offspring. Our results demonstrate that generating hu- species, especially in small colonies, and this could be achieved man or domestic animal offspring from space-preserved spermatozoa is a possibility, which should be useful when the by cryopreserving a diverse range of gamete cells. The environ- “ ” ment in space is very different from that on Earth, however, space age arrives. including high levels of space radiation and microgravity, and the Author contributions: S.W., T.K., H.S., T.S., F.I., S.Y., and T.W. designed research; S.W., effects of these factors on mammalian reproduction are largely Y.K., K.Y., T.K., H.S., T.S., M.N.T., I.O., A.N., S.K., H.N., E.M., S.Y., and T.W. performed unknown. Although with current technology, producing offspring research; T.K., A.N., and F.I. analyzed data; and S.W. and T.W. wrote the paper. in such an environment can be difficult or dangerous (5), the The authors declare no conflict of interest. study of reproduction in space is a very important subject for This article is a PNAS Direct Submission. our future. Freely available online through the PNAS open access option. So far, the effects of microgravity on early development have Data deposition: Sequences have been deposited in the DNA Data Bank of Japan Se- been studied using sea urchins, fish, amphibians, and birds (6– quence Read Archive (DRA submission 005694). 12). These studies have concluded that microgravity does not 1To whom correspondence may be addressed: Email: [email protected] or prevent animal reproduction. However, because of the difficulty [email protected]. in maintaining mammals and performing experiments in space, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. studies of mammal reproduction in space have not progressed as 1073/pnas.1701425114/-/DCSupplemental. 5988–5993 | PNAS | June 6, 2017 | vol. 114 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1701425114 Downloaded by guest on September 25, 2021 repairing damaged DNA (25–27), so when live animals are in a small volume. Therefore, our samples could be launched to the space for only short periods, the effect of space radiation might ISS without the need for a freezer, which greatly reduced the cost be masked by subsequent repair of the damaged DNA after of launching. The merits of this procedure, in terms of the ease of fertilization. In contrast, although cryopreserved cells are still launching freeze-dried samples into space, are significant for en- alive, they have stopped metabolizing, which means that they abling the study of mammalian reproduction in space, even though cannot repair DNA damage while still frozen (28, 29). There- the production rate of offspring from freeze-dried spermatozoa is fore, DNA damage might accumulate with increased duration in lower than that from traditional cryopreservation methods. space. If this is so, then the production of offspring from long- After exposure to space radiation for 9 mo at –95 °C, the term cryopreserved spermatozoa in space will be compromised samples were returned to Earth. We evaluated these samples for because of increased embryo mortality or mutation rates in the sperm morphology and DNA damage, capacity for fertilization offspring. Therefore, it is very important to examine the effect of by microinjection, in vitro developmental potential, and nor- space radiation on spermatozoa preserved in space. In addition, mality of offspring derived from the spermatozoa. the resistance of spermatozoa likely differs among species, because the sperm structure differs (30). Therefore, to examine the Results influences of radiation in mammalian species, we must use Collection of Space Sperm Samples. The samples (Fig. 1 A and B) mammalian species, and the mouse is a very convenient model were launched to the ISS on August 4, 2013, and returned to animal for space study. ground on May 19, 2014. Therefore, these space sperm samples For the present study, we decided to freeze-dry the mouse were exposed to cosmic radiation for 288 d. The majority of the spermatozoa (31) rather than use traditional cryopreservation glass ampules sustained no damage during the launch or return. methods. When spermatozoa are freeze-dried or evaporatively The ground control sperm samples from the same mice were dried, none of the sperm survive; however, mouse spermatozoa exposed to the same temperature changes at the same times and can maintain the ability to generate offspring when added to for the same durations as the space sperm samples. water and microinjected into fresh oocytes (31, 32), and this may possible with human spermatozoa in the future (33, 34). More Total Doses of Space Radiation. The CR-39 plastic nuclear track importantly, such dried spermatozoa can be preserved at room detectors (PNTDs) in the Bio PADLES packages collected from temperature for up to 2 y (35, 36) and in a freezer almost in- the space-preserved cases (Fig.

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